Chemoenzymatic preparation of enantiopure l-benzofuranyl- and l-benzo[b]thiophenyl alanines

Article abstract of DOI:10.1016/j.tetasy.2008.01.031

Lipase mediated DKR followed by a chemical and an enzymatic hydrolytic step were combined for the synthesis of enantiopure l-benzofuranyl- and l-benzothienyl alanines.

Full text of DOI:10.1016/j.tetasy.2008.01.031

Available online at www.sciencedirect.com
Tetrahedron: Asymmetry 19 (2008) 500–511
Chemoenzymatic preparation of enantiopure L-benzofuranyl- and
L-benzo[b]thiophenyl alanines
Paula Veronica Podea, Monica Ioana Tosa, Csaba Paizs and Florin Dan Irimie*
ß
Department of Biochemistry and Biochemical Engineering, Babeßs-Bolyai University, 400028-Arany Ja´nos 11, Cluj-Napoca, Romania
Received 13 December 2007; accepted 23 January 2008
Abstract—Lipase mediated DKR followed by a chemical and an enzymatic hydrolytic step were combined for the synthesis of enantio-
pure L-benzofuranyl- and L-benzothienyl alanines.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
backs of traditional resolution [maximum 50% theoretical
yield and the decrease of enantiomeric excess (ee) with
the conversion] can be overcome by dynamic kinetic reso-
lution (DKR), whereby a racemic mixture is transformed
to one of the enantiomers by in situ racemizing the less
reactive enantiomer in the course of the process.
Both naturally occurring and unnatural a-arylalanines are
frequent constituents of biological and pharmaceutical
products. Several non-proteinogenic aminoacids as L-
Dopa, L-homophenylalanine or D-2-naphthylalanine are
already important pharmaceuticals,1 while others such as
D-phenylglycine, D-para-hydroxy-phenylglycine, and 2-thi-
enyl-alanine are useful for the synthesis of certain drugs.2
Many others serve as building blocks for modern drug dis-
covery research.3 The enantioselective synthesis of amino
acids is an attractive goal. While L-natural aminoacids
are obtained by fermentation or by applying natural enzy-
matic systems, for enantiopure unnatural aminoacid syn-
thesis, chiral chemical catalysts or biocatalysts must be
used. The use of ammonia lyases4,5 or oxidoreductases1
for the enantioselective asymmetrization of prochiral sub-
strates provides the desired enantiopure amino acids with
100% theoretical yield. However, their use for biocatalysis
is rather limited, as they show not only a strict reaction and
enantioselectivity but are also specific for one or a few sub-
strates. Therefore, enzymes with broad substrate selectiv-
ity, while maintaining other selectivities are the preferred
biocatalysts. Hydrolases (acylases,6 amidases7 and hydan-
toinases, esterases,8 nitrilases or nitrilhydratases9) fulfil
these criteria and by kinetic resolution of different kinds
of racemic substrates are the most important biocatalysts
for enantioselective aminoacid synthesis. The main draw-
Under such conditions, the starting material is always race-
mic, allowing the maximum enantiopurity (determined by
the E value for the corresponding kinetic resolution) at
the theoretical zero conversion to the more reactive enan-
tiomer to be maintained throughout the reaction. This is
extremely important for an enzymatic reaction proceeding
enantioselectively, since the ee of the product enantiomer
tends to decrease with increasing conversion of kinetic res-
olution especially at E values less than 100.
It was shown that 2-substituted oxazol-5(4H)-ones, due to
the low pKa of the C-4 proton10 and their high reactivity
with different nucleophiles, are excellent substrates for
enzymatic dynamic kinetic resolution. The enzymatic
DKR of 4-substituted 2-phenyloxazol-5(4H)-one proved
to be an efficient and elegant method for enantiopure L-
phenyl-alanine11 and L-tert-leucine synthesis.12
Herein, we report the chemoenzymatic synthesis of enan-
tiopure L-2-amino-3-(heteroaryl)propanoic acids starting
from the corresponding racemic 2-acetamido-3-(hetero-
aryl)propanoic acids. Enantiopure L-aminoacids (ee
>99%) were produced with high yields (ꢀ80%) combining
the enzymatic DKR of the corresponding oxazolones with
a chemical and an enzymatic hydrolytic step.
*
Corresponding author. Tel.: +40 264 593833; fax: +40 264 590818;
e-mail: irimie@chem.ubbcluj.ro
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.01.031

P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
501
2. Results and discussion
2.1. Chemical synthesis
were synthesized by the reduction of the aldehydes with
NaBH4 in methanol, while halogenated compounds 6a–c
were prepared with thionylchloride in dichloromethane in
the presence of 1H-benzo[d][1,2,3]triazole. The use of 1H-
benzo[d][1,2,3]triazole overcame the destruction of acid-
sensitive compounds, enhancing the yields of the reactions.
The coupling of the halogenated compounds 6a–d with
diethyl-2-acetamido-malonate afforded the diethyl-2-acet-
amido-2-((heteroaryl)methyl)malonate 7a–d. By mild basic
hydrolysis of the diethyl esters formed, 2-acetamido-2-
((heteroaryl)methyl)malonic acids 8a–d were obtained,
which were further decarboxylated in boiling toluene. In
the final step, 2-acetamido-3-(heteroaryl)propanoic acids
rac-9a–d were chemically transformed into the racemates
involved in the stereoselective enzymatic reactions. Race-
mic 9a–d were transformed with alcohols in dry tetrahydro-
furan in the presence of carbonyl diimidazole into 2-
acetamido-3-(heteroaryl)propanoic esters rac-10a–d; then
in the presence of dicyclohexyl carbodiimide (DCC)13 in
dry dichloromethane into the corresponding rac-4-((hetero-
aryl)methyl)-2-methyloxazol-5(4H)-ones rac-11a–d; the
protecting acetyl group of rac-9a–d was removed by acidic
hydrolysis and the phenylalanine analogues formed rac-
12a–d were isolated by precipitation at their isoelectric
point.
Commercially available benzofuran, benzo[b]thiophene
and 1-(2-hydroxyphenyl)ethanone were used as general
starting materials (Scheme 1).
The aldehyde group was selectively introduced at the 2-po-
sition of the benzofuran ring via a Vilsmeyer–Haack
formylation of benzofuran with phosphoryl trichloride in
DMF; however, benzofuran-2-carbaldehyde 4a was not
produced with high yield. The remaining benzofuran was
easily recovered because the quantity of the side products
was small. Benzo[b]thiophene was quantitatively trans-
formed into benzo[b]thiophen-2-yl-lithium with butyl lith-
ium at ꢁ78 °C, which was then easily transformed into
the corresponding aldehyde 4b with DMF.
The chloromethylation of benzo[b]thiophene takes place
selectively at the 3-position, but the yield was relatively
poor. The synthesis of benzofuran-3-carbaldehyde using
benzofuran as a starting material was unsuccessful. For
this reason, we chose a synthetic pathway which was based
on an intramolecular reaction, yielding a 3 substituted ben-
zofuran which could be transformed further into the de-
sired aldehyde. 1-(2-Hydroxyphenyl)ethanone was trans-
formed in (2-acetyl-phenoxy)acetic acid ethyl ester 1,
followed by the mild hydrolysis of the ester. The intramo-
lecular cyclisation of (2-acetyl-phenoxy)acetic acid 2 fol-
lowed by the decarboxylation of the intermediate allowed
us to isolate 3-methyl-benzofuran 3 by in vacuo distillation.
By the oxidation of the latest compound with SeO2, benzo-
furan-3-carbaldehyde 4c was formed (Scheme 1).
2.2. Enzymatic synthesis
To investigate the stereoselectivity and the conditions of
DKR for the lipase mediated alcoholysis of 4-((hetero-
aryl)methyl)-2-methyloxazol-5(4H)-ones rac-11a–d, the
chromatographic separation of the enantiomers of the
reaction counterparts rac-4-((heteroaryl)methyl)-2-meth-
yloxazol-(4H)-ones rac-11a–d and rac-2-acetamido-3-(het-
eroaryl)propanoic esters rac-9a–d was first established
(Fig. 1a).
Aldehydes 4a–c and 3-(chloromethyl)benzo[b]thiophene 6d
were used as starting materials for the synthesis of racemic
amino acids and their derivatives (Scheme 2).
A large number of commercially available lipases exhibit a
high degree of substrate tolerance leading to products with
various degrees of enantioselectivity.
Aldehydes were then transformed into chloromethylene
derivatives via the corresponding alcohols. Alcohols 5a–c
O
O
II
I
S
S
O
O
4a
4b
O
O
O
O
O
III
IV
V
VI
O
OH
O
O
OH
O
O
O
1
2
3
4c
Cl
VII
S
S
6d
Scheme 1. Synthesis of heteroaryl aldehydes. Reagents and conditions: (I) POCl3/DMF; (II) (1) BuLi, ꢁ78 °C, 1 h, (2) DMF, ꢁ10 °C, 2 h; (III)
ClCH2COOEt, K2CO3/acetone; (IV) K2CO3/H2O; (V) Ac2O/AcOꢁNa+; (VI) SeO2/dioxane, reflux; (VII) CH2O/HCl, 60 °C.

502
P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
O
Cl
OH
EtOOC
R
HOOC
R
I
III
II
IV
VI
COOEt
COOH
R
R
R
NHCOCH3
7a-d
NHCOCH3
8a-d
4a-c
5a-c
6a-c
COOAlk
V
R
O
S
O
S
NHCOCH3
a
c
rac-10a-d
b
d
O
COOH
R
VII
R
R
O
N
NHCOCH3
rac-9a-d
rac-11a-d
COOH
VIII
NH2
rac-12a-d
Scheme 2. Synthesis of racemic heteroaryl alanines and their derivatives. Reagents and conditions: (I) NaBH4/CH3OH; (II) SOCl2, benzotriazole/CH2Cl2;
(III) NaH, CH3CONHCH(COOEt)2/DMF, 60 °C; (IV) 10% KOH, 20 h; (V) toluene/xylene, reflux, 2 h; (VI) alcohol, CDI/THF; (VII) DCC/CH2Cl2;
(VIII) 18% HCl, reflux, 4 h.
O
O
O
N
O
O
O
S
NHCOCH3
NHCOCH3
S
S
O
O
N
S
O
O
NHCOCH3
S
O
O
NHCOCH3
S
Figure 1. (a) Elution diagram of the mixture of the racemic starting material rac-11d and racemic product rac-10d for the lipase catalysed DKR.
(b) Elution diagram for the enzymatic DKR of rac-4-((benzo[b]thiophen-3-yl)methyl)-2-methyloxazol-5(4H)-one rac-11d: after 2 h (blue trace), after 24 h
(red trace) and after 48 h (green trace).
To obtain L-2-amino-3-(heteroaryl)propanoic esters L-10a–
d with good yields and high ee several enzymes were tested
as potential biocatalysts in neat ethanol as solvent and
nucleophile. All lipases, except for lipase F, showed the

P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
503
same enantiopreference in their alcoholysis, although the
behaviour of lipases greatly differed. Lipases AY, PS,
CcL and CrL were catalytically inactive; lipase AK (ee
13–17%), lipase F (ee 5–7%) and Lipozyme TL IM (ee 7–
11%) gave moderate selectivity and reactivity, whereas
Novozyme 435 showed acceptable properties (Table 1,
entry 2). Accordingly, Novozyme 435 was used for further
studies.
methyloxazol-5(4H)-ones rac-11a–d (Table 2, entry 1). It
was previously1,11 shown that the conversion of the reac-
tion and the ee of the products in the enzymatic dynamic
kinetic resolution of rac-4-substituted 2-phenyloxazol-
5(4H)-ones were strongly influenced by the presence of
water and triethylamine and by the nucleophile substrate
and the enzyme–substrate ratio. It is worth emphasizing
that even small quantities of water caused a decrease in
the ee of the enzymatic alcoholysis product. The generated
carboxylic acid most likely interacts with the enzyme lead-
ing to a change in the conformation that alters the stereo-
complementarity between enzyme and the reacting enantio-
mer of the substrate. To avoid this inconvenience, all enzy-
matic reactions were carried out in anhydrous conditions.
It should be noted that before use, the enzymes were dried
or crashed and dried (in case of immobilized enzymes), as
described by Turner et al.11 In contrast to all earlier re-
ported results, rac-4-((heteroaryl)methyl)-2-methyloxazol-
5(4H)-ones rac-11a–d showed spontaneous racemization
during the enzymatic reaction (Fig. 1b, blue trace and red
trace); the presence of triethylamine in the reaction media
was not helpful. Moreover, by favouring the non-enzy-
matic alcoholysis, even a very small quantity of triethyl-
amine (0.1 equiv) increased the reaction time and
It is known that the nature of the nucleophile and solvent
could substantially influence the selectivity of the enzy-
matic reaction. For Novozyme 435 mediated alcoholysis
of rac-4-((heteroaryl)methyl)-2-methyloxazol-5(4H)-ones
rac-11a–d (Scheme 3a), two conditions were compared.
The reactions were first performed in alcohols at room tem-
perature. Four different alcohols were used as both solvent
and nucleophile.
1-Propanol was found to be the most selective nucleophilic
agent for all substrates (Table 1, entry 3). The enzymatic
propanolysis was carried out at room temperature in sev-
eral organic solvents.
Dioxane proved to be the most appropriate solvent for the
biocatalytic ring-opening of rac-4-((heteroaryl)methyl)-2-
Table 1. Enantiomeric excess for the Novozyme 435 mediated alcoholysis
of rac-4-((heteroaryl)methyl)-2-methyloxazol-5(4H)-ones rac-11a–d
Table 2. Enantiomeric excess for DKR of rac-4-((heteroaryl)methyl)-2-
methyloxazol-5(4H)-ones rac-11a–d in organic solvents
Entry
Type of ester
ee for L-2-amino-3-
(heteroaryl)propanoic estersa,b (%)
Entry
Solvent
ee for L-2-amino-3-(heteroaryl)-
propanoic acid propyl esters (%)
L-10a
L-10b
L-10c
L-10d
L-10a
L-10b
L-10c
L-10d
1
2
3
4
Methyl ester
Ethyl ester
Propyl ester
Butyl ester
3
67
71
67
3
65
71
69
3
65
69
67
3
73
79
75
1
2
3
4
5
6
Dioxane
Dichloromethane
Toluene
Acetonitrile
Tetrahydrofuran
Diethylether
75
39
69
61
71
67
79
37
59
55
47
71
83
42
69
73
81
59
86
54
79
83
83
73
a In neat alcohol.
b Total conversion after 6 h.
O
COOH
COOH
NH2
L-12a-d
COOH
Acylase I
pH 7-8
R
R
R
b
+
R
O
NHCOCH3
NHCOCH3
N
(rac)-9a-d
D-9a-d
O
S
D-11a-d
a
b
O
O
COOPr
NHCOCH3
COOH
NHCOCH3
III
II
R
R
R
a
N
O
S
L-9a-d (ee 81-87%)
L-10a-d (ee 81-87%)
(rac)-11a-d
c
d
c
IV
I
O
COOAlk
COOH
R
COOH
NHCOCH3
(rac)-9a-d
Lipase
R
O
N
R
R
Alcohol
NHCOCH3
L-10a-d
NH2
L-11a-d
L-12a-d (ee>99%)
Scheme 3. Enantioselective synthesis of L-heteroaryl alanines and their derivatives: (a) DKR of the lipase mediated alcoholysis of oxazolones rac-11a–d;
(b) enantiomer selective hydrolysis of rac-9a–d; (c) chemoenzymatic synthesis of enantiopure L-12a–d. Reagents and conditions: (I) DCC/CH2Cl2; (II)
propanol, Novozyme 435/dioxane; (III) Na2CO3, H2O, reflux; (IV) Acylase I, pH 7–8.

504
P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
decreased the enantioselectivity of the global transforma-
tion. A high nucleophile–substrate ratio (3–10) also de-
creased the stereoselectivity of the lipase mediated
reaction. It was found that 2 equiv of the nucleophile was
the optimal amount necessary for total conversion of
rac-4-((heteroaryl)methyl)-2-methyloxazol-5(4H)-ones rac-
11a–d (Fig. 1b, green trace), while the concurrent
non-enzymatic alcoholysis decreased the ee when the
substrate–enzyme ratio was higher than 1:1 (m/m). These
results are in good agreement with our previous observa-
tion concerning the non-enzymatic alcoholysis of
the rac-4-((heteroaryl)methyl)-2-methyloxazol-5(4H)-ones.
It was found that in neat alcohols rac-4-((heteroaryl)-
methyl)-2-methyloxazol-5(4H)-ones rac-11a–d were totally
transformed into racemic products in about 1 day, while
using triethylamine as a catalyst (0.25 equiv) total conver-
sion of the substrate occurs in 5–6 h.
mAU
250
200
150
100
50
COOH
COOH
NHCOCH
3
NHCOCH
3
S
S
0
0
2
4
6
8
10
12
14
16
min
Figure 2. Elution diagram of the isolated product of the chemical
hydrolysis, L-2-acetamido-3-(benzo[b]thiophene-3-yl)propanoic acid L-9d
(red trace) and rac-2-acetamido-3-(benzo[b]thiophene-3-yl)propanoic acid
rac-9d as reference (blue trace).
Starting from the enantiomerically enriched L-2-acet-
amido-3-(heteroaryl)propanoic acids propyl esters L-10a–
d, a second chiral selector had to be used in one of the next
two hydrolytic steps, to obtain enantiopure L-2-amino-3-
(heteroaryl)propanoic acids. Acylase I proved to be a
stereospecific catalyst for the kinetic resolution of racemic
The expected L-configuration of all heteroaryl alanines was
confirmed by measuring their specific rotations, which were
consistent with the literature values.
3. Conclusions
2-acetamido-3-(heteroaryl)propanoic
acids
rac-9a–d
(Scheme 3b). Enantiopure L-heteroaryl-amino acids
L-12a–d and D-heteroaryl-N-acetyl-amino-acids D-9a–d
were isolated from the reaction mixture.
The present work describes the usability of a chemo-
enzymatic procedure for the preparation of enantiopure
heteroaryl alanines starting from racemic 2-acetamido-3-
(hetero- aryl)propanoic acids. The first enzyme catalyzed
step was the Novozyme 435 mediated dynamic kinetic res-
olution of the oxazolones which provided the N- and C-
protected L-amino acids (ee 76–85%) with theoretical 100%
yield. The protecting groups were removed with excellent
yields by combining a second chemical (mild hydrolysis
of the ester group) and a third enzymatic step (hydrolysis
of the amide group). The latest, due to the L-specificity of
Acylase I, raised the ee of the final product to more than
99%.
With these results in hand, the preparative scale synthesis
of the enantiopure L-12a–d from racemic 2-acetamido-3-
(heteroaryl)propanoic acids rac-9a–d was set up (Scheme
3c).
The progress of the reaction was monitored after each step
by HPLC to determine the enantiopurity of the isolated
compounds and the conditions for quantitative conver-
sions. The lipase mediated DKR was stopped when the
consumption of the rac-4-((heteroaryl)methyl)-2-meth-
yloxazol 5(4H)-ones rac-11a–d ceased. The ee of the iso-
lated products was the same as the one found in the case
of small scale reactions. Then, by the chemical hydrolysis,
without altering the ee (Fig. 2), the enantiomer enriched
L-2-acetamido-3-(heteroaryl)propanoic acid propyl esters
L-10a–d were transformed in good yields (ꢀ98%)
into L-2-acetamido-3-(heteroaryl)-propanoic acids L-9a–d.
4. Experimental
4.1. Analytical methods
1
The H and 13C NMR spectra were recorded on a Bruker
spectrometer operating at 300 MHz and 75 MHz, respec-
tively, at 25 °C. Electron impact mass spectra (EI-MS)
were taken on a VG 7070E mass spectrometer operating
at 70 eV.
After the enantiospecific Acylase I mediated hydrolysis of
the latest compounds, the enantiopure final products,
L-heteroarylalanines L-12a–d were easily isolated on an
ion-exchange column. The yields and the specific rotation
values of the isolated final products are shown in
Table 3.
High performance liquid chromatography (HPLC) analy-
ses were conducted with a HP 1200 instrument using an
Astec Chirobiotic-Tag column (4.6 ꢂ 250 mm) and a mix-
ture of methanol and TEAA-buffer (pH 4.1), 80:20 (v/v)
as eluent for the enantiomeric separation of rac-9,12a–d
and a Chiralpak IA column (4.6 ꢂ 250 mm) and a mixture
of hexane and 2-propanol, 90:10 (v/v) as eluent for enan-
tiomeric separation of rac-10–11a–d, both at 1 mL/min
flow rate. For all chiral compounds, high resolution enan-
tiomeric separation was performed. Retention times for L-
and D-9–12a–d are presented in Table 4.
Besides the yields, which varied between 76% and 85%, the
absolute configurations and the enantiomeric purity of the
isolated heteroarylalanines were also determined. Figure 3
shows the elution diagram on a chiral column of the
L-2-amino-3-(benzo[b]thiophene-3-yl)propanoic acid L-12d
(red trace) produced and that of the racemic 2-amino-
3-(benzo[b]thiophene-3-yl)propanoic acid rac-12d (blue
trace) as a control.

P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
Table 3. Yields and specific rotations for the isolated enantiopure L-amino acids L-12a–d
505
20
Substrate
Producta
Yieldb (%)
½aꢃD (10 mg mLꢁ1
)
rac-9a
rac-9b
rac-9c
rac-9d
L-12a
L-12b
L-12c
L-12d
76
79
83
85
ꢁ14.5, CH3COOH (ꢁ14.5, CH3COOH, 20 °C5)
ꢁ23.8, CH3COOH (ꢁ23.8, CH3COOH, 20 °C5)
ꢁ8.0, CH3COOH
ꢁ26.0, CH3COOH (ꢁ9.2, 0.1 M HCl, 20 °C14
)
a ee >99% for all the compounds.
b Yields are given for the isolated compounds.
4.2. Reagents and solvents
mAU
600
Benzofuran, benzo[b]thiophene, 1-(2-hydroxyphenyl)-etha-
none, ethyl 2-chloroacetate, all inorganic reagents and sol-
vents were produced from Aldrich or Fluka. All solvents
were purified and dried by standard methods as required.
Lipases AY, AK, PS and F were from Amano, Europe,
England. Lipases from Candida rugosa (CrL), Candida
cylindracea (CcL) and Acylase I were purchased from
Fluka. Lipase B (CAL-B, Novozyme 435) from Candida
antarctica and Lypozyme TL IM were purchased from
Novozymes, Denmark.
500
400
300
COOH
COOH
NH
2
NH
2
S
S
200
100
0
0
2.5
5
7.5
10 12.5 15 17.5
20 min
Figure 3. Elution diagram of the isolated enantiopure L-2-amino-3-
(benzo[b]thiophene-3-yl)propanoic acid L-12d (red trace) and rac-2-
amino-3-(benzo[b]thiophene-3-yl)propanoic acid rac-12d as reference
(blue trace).
4.3. Chemical synthesis of racemic heteroaryl alanines and
their derivatives
4.3.1. Synthesis of benzofuran-2-carbaldehyde 4a. Phos-
phoryl trichloride (132 g, 80 mL, 0.86 mol) was added in
small portions with slight warming into a solution of benzo-
furan (92 g, 0.78 mol) in anhydrous dimethylformamide
(150 g, 158 mL, 2.05 mol). The mixture was heated for
10 h at 100 °C, then cooled to room temperature and trea-
ted with an extra portion of dimethylformamide (50 g,
52.7 mL, 0.68 mol) and phosphoryl trichloride (40 g,
24.3 mL, 0.26 mol). The mixture was heated for another
10 h. The cooled mixture was treated with saturated
sodium acetate solution in water until the pH rose to 6.
The organic compounds were extracted with Et2O
(3 ꢂ 100 mL). The combined organic layers were washed
with saturated KHCO3 solution (3 ꢂ 50 mL) and water
(100 mL), and finally dried over anhydrous MgSO4. The
solvent was removed and the residue distilled in vacuo.
Yield = 62%; bp 135–136 °C/18 mmHg (135–136 °C/
18 mmHg15); HRMS: M+ found (M+ calculated for
C9H6O2): 146.03701 (146.03678); MS: m/z (%) = 148
(1.53, M+2), 147 (19.73, M+1), 146 (100, M), 145
(87.96), 138 (0.44), 131 (0.36), 121 (0.29), 120 (0.71), 119
(1.18), 118 (15.48), 117 (0.68), 116 (0.39), 102 (0.40), 101
Table 4. Retention times of the enantiomers of rac-9–12a–d
rt (min)
L-9a
D-9a
L-9b
D-9b
L-9c
D-9c
L-9d
D-9d
2.8
11.6
2.9
8.7
2.8
11.0
3.0
13.1
D-10a L-10a
D-10b L-10b
D-10c L-10c
D-10d L-10d
12.5a
10.4b
10.6c
9.5d
14.4a
12.2b
12.6c
11.0d
12.0a
9.3b
11.5c
10.2d
13.6a
12.3b
15.8c
13.2d
8.1a
11.2b
11.5c
11.2d
10.9a
12.9b
13.0c
12.9d
12.0a
10.5b
10.7c
9.4d
15.0a
13.5b
14.0c
11.8d
11a
11b
11c
11d
8.9
12.3
11.3
11.5
7.5
8.1
8.2
9.0
L-12a
D-12a L-12b
14.9 10.1
D-12b L-12c
15.2 8.9
D-12c L-12d
13.5 10.5
D-12d
8.8
15.7
a Methyl ester.
b Ethyl ester.
c Propyl ester.
d Butyl ester.
1
(1.13), 98 (0.33), 92 (0.64); H NMR (CDCl3): d = 7.24–
7.28 (m, 1H), 7.42–7.46 (m, 1H), 7.51 (s, 1H), 7.52 (d,
1H), 7.67 (d, 1H), 9.79 (s, 1H); 13C NMR (CDCl3):
d = 112.7, 117.9, 123.7, 124.2, 126.7, 129.3, 152.7, 156.3,
179.8.
Thin layer chromatography (TLC) was carried out using
Merck Kieselgel 60F254 sheets. Spots were visualized by
treatment with 5% ethanolic phosphomolybdic acid solu-
tion and heating. Preparative chromatographic separations
were performed using column chromatography on Merck
Kieselgel 60 (63–200 lm). Melting points were determined
by a hot plate method and are uncorrected. Optical rota-
4.3.2. Synthesis of benzo[b]thiophene-2-carbaldehyde
4b. Into a cooled solution (ꢁ78 °C) of benzo[b]thiophene
(3.24 g, 2.17 mL, 20 mmol) in anhydrous THF (120 mL),
2.7 M n-BuLi in heptane (ꢀ8.15 mL, 1.1 equiv) was added
dropwise under nitrogen, and the mixture was stirred for
1 h at ꢁ78 °C. Then, anhydrous DMF (4.38 g, 4.62 mL,
60 mmol) was added and the solution was stirred for 2 h
tions were determined on a Perkin–Elmer 201 polarimeter
20
and ½aꢃD values are given in units of 10ꢁ1 deg cm2 gꢁ1
.

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P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
1
while warming the reaction mixture to ꢁ10 °C. The mix-
ture was hydrolyzed with saturated NH4Cl solution
(30 mL) and diluted with water (100 mL). After the separa-
tion of the phases, the water phase was extracted with Et2O
(3 ꢂ 50 mL). The combined organic layers were washed
with water and brine, dried over anhydrous MgSO4 and
concentrated in vacuo. The crude product was purified by
column chromatography on silica gel using dichlorometh-
ane as eluent. Yield = 62%; mp 34–35 °C from ethanol
(34–34.3 °C from ethanol16) HRMS: M+ found (M+ calcu-
lated for C9H6OS): 162.01418 (162.01394); MS: m/z
(%) = 164 (15.42, M+2), 163 (56.40, M+1), 162 (100, M),
161 (97.75), 147 (0.89), 136 (2.06), 135 (6.49), 134 (58.68),
133 (61.29), 132 (5.52), 108 (5.84), 107 (1.25), 106 (3.02),
105 (1.69), 102 (2.94), 98 (1.58); 1H NMR (CDCl3):
d = 7.34–7.43 (m, 1H), 7.43–745 (m, 1H), 7.82 (d, 1H),
7.87 (d, 1H), 7.95 (s, 1H), 10.03 (s, 1H); 13C NMR
(CDCl3): d = 122.3, 124.3, 125.3, 127.2, 133.5, 137.5,
141.7, 142.3, 183.7.
(1.75); H NMR (CDCl3): d = 2.61 (s, 3H), 4.71 (s, 2H),
6.89 (d, 1H), 7.04–7.08 (m, 1H), 7.44–7.48 (m, 1H), 7.72
(d, 1H); 13C NMR (CDCl3): d = 29.1, 65.8, 113.7, 121.5,
126.5, 130.3, 133.6, 155.8, 169.8, 199.5.
4.3.5. Synthesis of benzofuran-3-carbaldehyde 4c. A mix-
ture of (2-acetyl-phenoxy)acetic acid (77.5 g, 0.40 mol)
and anhydrous sodium acetate (141 g, 1.7 mol) in acetic
anhydride (285 g, 2.8 mol) was heated at 160 °C for 3 h.
After cooling, the mixture was poured into 900 mL water
and then extracted with Et2O (3 ꢂ 300 mL). The combined
organic layers were washed with 10% Na2CO3 solution
(3 ꢂ 150 mL) and then with water (3 ꢂ 100 mL) and finally
dried over MgSO4. The solvent was removed and 3-methyl-
benzofuran was isolated by distillation. Then a mixture of
3-methyl-benzofuran (10.02 g, 75.8 mmol) and selenium
dioxide (9.70 g, 87.4 mmol) in dry 1,4-dioxane (100 mL)
was refluxed for 48 h. The resulting black precipitate was
filtered off and the crude product fractionated by distilla-
tion under reduced pressure.Yield = 77%; bp 69 °C/
0.4 mmHg (68–69 °C/0.4 mmHg18); HRMS: M+ found
(M+ calculated for C9H8O2): 146.03705 (146.03678); MS:
m/z (%) = 148 (0.62, M+2), 147 (6.48, M+1), 146 (82.02,
M), 145 (100), 144 (0.15), 136 (0.20), 131 (0.15), 121
(0.63), 119 (0.15), 118 (1.52), 117 (1.41), 116 (0.16), 109
(0.14), 107 (0.28), 106 (0.16), 105 (0.19), 102 (0.16), 101
(0.26), 98 (0.14); 1H NMR (CDCl3): d = 7.27–7.34 (m,
2H), 7.45 (d, 1H), 8.10 (d, 1H), 8.17 (s, 1H), 10.07 (s,
1H); 13C NMR (CDCl3): d = 111.7, 122.6, 122.9, 123.7,
124.9, 126.3, 155.4, 156.0, 184.8.
4.3.3. Synthesis of (2-acetyl-phenoxy)acetic acid ethyl ester
1. A mixture of 1-(2-hydroxyphenyl)ethanone (110 g,
97.26 mL, 0.80 mol), ethyl 2-chloroacetate (122.5 g,
107 mL, 1.0 mol) and anhydrous K2CO3 (110.5 g,
0.80 mol) in acetone (600 mL) was refluxed for 58 h. After
cooling, the precipitate was filtered off and washed with
acetone (3 ꢂ 150 mL). The acetone was removed by distil-
lation from the combined solutions and the crude product
was distilled under reduced pressure affording 1. Yield:
58%, bp 185 °C/5 mmHg (185 °C/5 mmHg17); HRMS:
M+ found (M+ calculated for C12H14O4): 222.08889
(222.08921); MS: m/z (%) = 224 (1.14, M+2), 223 (9.67,
M+1), 222 (85.33, M), 208 (3.89), 207 (39.67), 204 (1.15),
193 (1.26), 180 (1.72), 179 (9.33), 177 (1.70), 176 (2.98),
153 (1.19), 152 (5.68), 151 (85.63), 150 (8.08), 149 (100),
148 (12.94), 147 (1.40), 137 (0.84), 136 (1.78), 135 (6.10),
134 (1.48), 133 (5.88), 132 (1.10), 131 (5.12), 124 (0.76),
123 (9.80), 122 (4.17), 121 (62.24), 120 (7.44), 119 (1.31),
118 (1.25), 107 (5.43), 106 (2.13), 105 (26.11), 104 (1.70),
103 (1.91), 101 (3.35), 95 (1.13); 1H NMR (CDCl3):
d = 1.23 (t, 3H), 2.65 (s, 3H), 4.21 (q, 2H), 4.65 (s, 2H),
6.75 (d, 1H), 6–6.99 (m, 1H), 7.35–7.39 (m, 1H), 7.69 (d,
1H); 13C NMR (CDCl3): d = 13.1, 31.0, 60.5, 64.3, 111.2,
120.7, 127.8, 129.7, 132.5, 155.9, 167.1, 198.7.
4.3.6. Synthesis of 3-chloromethyl-benzo[b]thiophene 6d.
A
rapid stream of HCl gas was passed into a vigorously
stirred mixture of 37% formaldehyde (9.7 g, 0.12 mol),
concentrated hydrochloric acid (9.7 mL, 0.11 mol) and
benzo[b]thiophene (13.05 g, 0.97 mol) until the mixture
was saturated with HCl. During this time, the temperature
of the mixture rose to 65 °C. This temperature was main-
tained for 1 h, while a slow stream of hydrogen chloride
gas was passed into the mixture. After the reaction was
completed, water (100 mL) was added to the cold reaction
mixture, the organic layer was separated and the water
phase extracted with Et2O (3 ꢂ 50 mL). The combined
organic layers were washed successively with water
(3 ꢂ 50 mL), saturated Na2CO3 solution (3 ꢂ 25 mL) and
finally with water (3 ꢂ 50 mL). The organic layer was dried
over MgSO4, and then the ether was evaporated. The crude
product was fractionated in vacuo. Yield 74.3%; bp 125–
127 °C/2 mmHg (125–127 °C/2 mmHg19).
4.3.4. Synthesis of (2-acetyl-phenoxy)acetic acid 2. Into
the vigorously stirred solution of sodium carbonate (53 g,
0.50 mol) in water (800 mL), (2-acetyl-phenoxy)acetic acid
ethyl ester (100 g, 0.45 mol) was added and the mixture was
gently refluxed. After 1 h of heating the solution was
cooled to 5 °C and acidified carefully with concentrated
HCl solution. The deposited precipitate was filtered off,
washed several times with cold water. Yield = 97%; mp
115 °C from water (114–114.3 °C from water16); HRMS:
M+ found (M+ calculated for C10H10O4): 194.05708
(194.05791); MS: m/z (%) = 195 (1.42, M+1), 194 (12.32,
M), 180 (0.76), 179 (7.63), 176 (0.92), 153 (5.12), 152
(2.66), 151 (25.10), 150 (33.02), 149 (6.50), 148 (6.88), 137
(1.06), 136 (11.71), 135 (56.58), 134 (1.45), 133 (7.52), 132
(3.50), 131 (2.83), 124 (0.89), 123 (8.21), 122 (7.53), 121
(100), 120 (4.40), 119 (0.84), 118 (1.01), 108 (1.58), 107
(10.66), 106 (3.26), 105 (18.51), 104 (2.74), 103 (1.26), 95
4.3.7. Reduction of aldehydes 4a–c. To a stirred solution
of aldehyde 5a–c (4 g) in methanol (40 mL), NaBH4 was
added in small portions at room temperature, until the en-
tire amount of the aldehyde was transformed. The progress
of the reaction was followed by thin layer chromatography,
using dichloromethane as eluent. Then the reaction mixture
was treated with 1M HCl (4 mL) and the methanol was re-
moved in vacuo. The crude product was treated with a mix-
ture of dichloromethane–water = 1:1 (v/v), the organic
layer was separated, dried over anhydrous MgSO4 and
the dichloromethane was removed in vacuo. The crude
product was purified by column chromatography, using

P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
507
dichloromethane–acetone = 9:1 (v/v) as eluent. The pure
compound was immediately used in the next step.
363.11375 (363.11404); MS: m/z (%) = 365 (3.41, M+2),
364 (10.40, M+1), 363 (51.63, M), 319 (1.00), 318 (5.38),
307 (2.19), 306 (13.20), 305 (45.85), 304 (1.00), 290 (1.49),
276 (1.76), 260 (1.21), 259 (6.45), 249 (2.46), 248 (17.11),
247 (3.26), 244 (4.34), 233 (1.75), 232 (12.49), 231 (5.42),
230 (27.91), 229 (2.64), 219 (2.66), 216 (1.21), 215 (3.15),
214 (21.84), 204 (1.51), 203 (1.38), 202 (3.75), 188 (1.28),
187 (4.31), 186 (13.03), 176 (4.45), 175 (13.91), 174
(34.85), 173 (8.01), 172 (2.73), 163 (1.06), 162 (1.07), 161
(1.41), 160 (2.70), 159 (2.85), 158 (11.70), 149 (4.96), 148
(10.01), 147 (61.04), 146 (4.71), 145 (2.30), 134 (3.04), 118
(1.25), 115 (4.37), 103 (2.73), 102 (1.74), 89 (1.16); 1H
NMR (CDCl3): d = 1.24 (t, 6H), 1.93 (s, 3H), 3.81 (s,
2H), 4.24 (q, 4H), 6.36 (s, 1H), 6.66 (s, 1H, NH), 7.08–
7.16 (m, 2H), 7.26 (d, 1H), 7.40 (d, 1H); 13C NMR
(CDCl3): d = 14.0, 23.0, 31.9, 63.0, 65.6, 105.8, 110.9,
120.7, 122.7, 123.9, 128.3, 153.0, 154.9, 167.2, 169.4.
4.3.8. Synthesis of chloromethylene derivatives 6a–c.
A
stock solution (1.5 M) was prepared by mixing up a volume
of a viscous clear solution of thionyl chloride (5.46 mL,
0.075 mol) and 1H-benzo[d][1,2,3]triazole (8.93 g, 0.075
mol) with dry dichloromethane up to 50 mL and trans-
ferred to a 50 mL burette. The reaction was started by add-
ing the stock solution (1.25 mmol equiv) intermittently
from the burette to a stirred solution of the alcohol 5a–c
(1 mmol) in dry dichloromethane (20 mL). Before the addi-
tion was complete, 1H-benzo[d][1,2,3]triazole hydrochlo-
ride started to precipitate. The reaction mixture was then
stirred further for 5–10 min. At the end the solid was fil-
tered off and washed with dry dichloromethane
(2 ꢂ 25 mL). The filtrate was washed with 10% HCl
(25 mL), water (25 mL), followed by 2% NaOH solution
(25 mL). The organic layer was separated and dried over
anhydrous MgSO4. The crude product was purified by dis-
tillation under reduced pressure and the distillate was
immediately used in the next step.
4.3.9.3. Diethyl 2-acetamido-2-((benzofuran-3-yl)-methyl)-
malonate 7c. Yield = 81%; mp 99 °C from ethanol;
HRMS: M+ found (M+ calculated for C18H21NO6):
347.13678 (347.13689); MS: m/z (%) = 348 (5.93, M+1),
347 (26.47, M), 302 (4.11), 289 (14.65), 288 (87.84), 259
(2.87), 243 (3.96), 242 (14.60), 232 (17.39), 228 (5.49), 215
(5.53), 214 (38.40), 213 (3.96), 198 (8.10), 186 (6.68), 185
(6.84), 174 (16.51), 171 (11.52), 170 (4.41), 160 (3.14), 159
(6.60), 158 (10.08), 157 (4.11), 153 (8.49), 149 (6.22), 146
(14.32), 136 (4.96), 132 (5.30), 131 (39.14), 125 (2.83), 115
(3.42), 111 (4.64), 107 (7.48), 106 (4.45), 105 (5.52), 103
4.3.9. Synthesis of malonic acid diethyl esters 7a–d. 55%
NaH oil suspension (0.85 g, 20 mmol) was added into dry
dimethylformamide (20 mL) and vigorously stirred for
30 min under argon at room temperature. Subsequently,
2-acetylamino-malonic acid diethyl ester (19.5 mmol) was
added to the suspension and then the reaction mixture
was stirred for 30 min. Chloromethylene derivative
(19 mmol) 6a–d was then added, after which the mixture
was stirred for 3 h at room temperature and for another
4 h at 60 °C. After cooling the reaction product was poured
into a water–ice mixture (100 g). The resulting precipitate
was filtered off, dried in vacuo at room temperature and fi-
nally purified by column chromatography on silica gel,
using as eluent, dichloromethane–CH3OH = 99:1 (v/v).
1
(7.18), 102 (4.31), 101 (5.98), 98 (3.65); H NMR (CDCl3):
d = 1.21 (t, 6H), 1.92 (s, 3H), 3.72 (s, 2H), 4.18 (q, 4H),
6.64 (s, 1H, NH), 7.11–7.15 (m, 1H), 7.17–7.21 (m, 1H),
7.25 (s, 1H), 7.34–7.37 (m, 2H); 13C NMR (CDCl3):
d = 14.0, 23.2, 26.5, 62.8, 66.9, 111.6, 114.2, 119.4, 122.6,
124.4, 128.4, 142.9, 155.0, 167.5, 169.4.
4.3.9.4. Diethyl 2-acetamido-2-((benzo[b]thiophen-3-yl)-
methyl)malonate 7d. Yield = 80%; mp 109–110 °C/from
ethanol (109–110 °C from benzene–petroleum ether20);
HRMS: M+ found (M+ calculated for C18H21NO5S):
363.11455 (363.11404); MS: m/z (%) = 364 (5.24, M+1),
363 (23.50, M), 306 (6.89), 305 (19.17), 304 (100), 259
(17.39), 256 (6.30), 248 (7.51), 230 (15.43), 228 (9.76), 214
(22.20), 191 (7.73), 187 (22.95), 186 (15.74), 182 (5.30),
178 (5.19), 174 (14.91), 158 (8.13), 154 (5.11), 153 (23.55),
149 (12.89), 148 (6.65), 147 (52.51), 140 (16.37), 136
(14.19), 125 (8.68), 115 (11.75), 112 (6.83), 111 (13.70),
108 (6.81), 107 (15.22), 106 (12.05), 105 (11.22), 101
(5.96), 98.1 (8.01); 1H NMR (CDCl3): d = 1.21 (t, 6H),
1.88 (s, 3H), 3.87 (s, 2H), 4.17 (q, 4H), 6.54 (s, 1H, NH),
7.22–7.29 (m, 2H), 7.60–7.64 (m, 1H), 7.73–7.77 (m, 1H);
13C NMR (CDCl3): d = 14.0, 23.1, 30.5, 62.9, 67.1, 121.7,
122.8, 124.0, 124.3, 124.4, 130.2, 139.5, 140.0, 167.5, 169.5.
4.3.9.1. Diethyl 2-acetamido-2-((benzofuran-2-yl)-methyl)-
malonate 7a. Yield = 80%; mp 93–94 °C from ethanol;
HRMS: M+ found (M+ calculated for C18H21NO6):
347.13130 (347.13689); MS: m/z (%) = 349 (1.36, M+2),
348 (9.56, M+1), 347 (49.88, M), 303 (1.73), 302 (10.36),
290 (7.84), 289 (54.75), 288 (100), 274 (3.22), 260 (3.89),
259 (1.60), 244 (3.02), 243 (16.04), 233 (3.95), 232 (36.13),
231 (6.49), 228 (8.13), 217 (3.64), 216 (32.49), 215 (7.72),
214 (16.25), 213 (4.26), 203 (6.80), 199 (2.32), 198 (9.85),
188 (4.90), 187 (2.46), 186 (5.86), 175 (3.70), 174 (23.31),
172 (1.44), 171 (7.01), 170 (15.81), 162 (1.52), 160 (3.94),
159 (12.16), 158 (16.07), 157 (14.83), 156 (1.98), 147
(3.28), 146 (22.91), 145 (1.53), 144 (3.43), 142 (6.83), 133
(1.58), 132 (7.82), 131 (66.22), 130 (3.55), 118 (5.04), 115
1
(3.73), 103 (4.25), 102 (3.67), 77 (7.62); H NMR (CDCl3):
d = 7.21 (t, 6H), 2.01 (s, 3H), 3.89 (s, 2H), 4.20 (q, 4H),
6.72 (s, 1H, NH), 6.89 (s, 1H), 7.17–7.25 (m, 1H), 7.59
(d, 1H), 7.67 (d, 1H); 13C NMR (CDCl3): d = 14.0, 23.2,
33.5, 63.0, 66.9, 122.1, 123.1, 124.1, 124.2, 124.3, 137.7,
139.5, 140.0, 167.1, 169.4.
4.3.10. Synthesis of rac-2-acetamido-3-(heteroaryl)-propa-
noic acids rac-9a–d. The diethyl 2-acetamido-2-((hetero-
aryl)methyl)malonate 7a–d (0.75 g) dissolved in methanol
(2.5 mL) was added in one portion into the 10% NaOH
solution (2.5 mL), after which the reaction mixture was
stirred for 20 h at room temperature. Subsequently, the
methanol was removed in vacuo and the pH of the solution
was adjusted to 1 with concentrated HCl, at ꢁ10 °C, giving
4.3.9.2. Diethyl 2-acetamido-2-((benzo[b]thiophen-2-yl)-
methyl)malonate 7b. Yield = 79%; mp 96 °C from etha-
nol; HRMS: M+ found (M+ calculated for C18H21NO5S):

508
P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
a white precipitate, which was filtered and dried in vacuo at
room temperature. The formed dicarboxylic acids 8a–d
were suspended in toluene (10 mL) and heated at reflux
for 2 h. The solvent was removed in vacuo, affording the
pure product.
4.3.10.4.
rac-2-Acetamido-3-(benzo[b]thiophen-3-yl)-
propanoic acid rac-9d. Yield = 85%; mp 154 °C from tol-
uene (153 °C from benzene–petroleum ether14); HRMS:
M+ found (M+ calculated for C13H13NO3S): 263.06180
(263.06161); MS: m/z (%) = 263 (22.57, M), 206 (5.38),
205 (9.72), 204 (80.60), 195 (3.92), 191 (4.19), 181 (4.36),
176 (4.69), 175 (4.37), 167 (5.28), 165 (4.75), 163 (4.22),
162 (7.43), 161 (4.47), 160 (4.29), 153 (7.79), 151 (6.44),
149 (10.58), 148 (10.67), 147 (100.00), 141 (4.07), 139
(4.49), 137 (7.13), 136 (4.06), 135 (4.72), 134 (4.88), 127
(4.87), 126 (4.02), 125 (10.91), 124 (4.15), 123 (8.97), 121
(4.98), 115 (7.52), 113 (5.17), 112 (4.43), 111 (16.32), 110
(5.23), 109 (11.35), 107 (4.75), 103 (4.43), 99 (6.05); 1H
NMR (DMSO-d6): d = 1.75 (s, 3H), 3.36–3.40 (m, 2H),
4.38–4.41 (m, 1H), 7.32–7.35 (m, 2H), 7.39 (s, 1H), 7.82
(d, 1H), 7.91 (d, 1H); 13C NMR (DMSO-d6): d = 22.7,
30.9, 53.9, 121.7, 122.6, 122.8, 123.8, 123.9, 133.6, 139.2,
139.3, 168.7, 175.5.
4.3.10.1. rac-2-Acetamido-3-(benzofuran-2-yl)propanoic
acid rac-9a. Yield = 81%; mp 115 °C from ethyl ace-
tate–hexane; HRMS: M+ found (M+ calculated for
C13H13NO4): 247.08482 (247.08446); MS: m/z (%) = 248
(2.12, M+1), 247 (14.31, M), 229 (0.51), 216 (0.63), 204
(1.88), 201 (0.67), 190 (1.08), 189 (11.04), 188 (100.00),
187 (0.74), 171 (2.41), 170 (0.56), 167 (0.55), 161 (0.91),
160 (7.32), 159 (2.84), 158 (0.63), 149 (1.02), 147 (2.51),
146 (5.42), 145 (0.62), 144 (2.93), 143 (0.60), 142 (1.30),
134 (0.74), 133 (1.42), 132 (7.61), 131 (52.11), 130 (1.02),
129 (0.46), 118 (1.63), 116 (0.54), 115 (2.74), 114 (0.46),
105 (1.22), 104 (0.63), 103 (3.11), 102 (2.04), 101 (0.55),
1
97.1 (0.60); H NMR (DMSO-d6): d = 1.80 (s, 1H), 3.05–
4.3.11. Synthesis of rac-propyl 2-acetamido-3-(hetero-
aryl)propanoates rac-10a–d. Into a solution of carbonyl-
diimidazole (90 mg, 0.55 mmol) and rac-2-acetamido-
3-(heteroaryl)propanoic acid rac-9a–d (0.5 mmol) in
anhydrous THF (2.5 mL), 1-propanol (45 mg, 56 lL, 0.75
mmol) was added in one portion at room temperature.
After the reaction was complete (checked by TLC), the sol-
vent was distilled off in vacuo and the crude product was
purified with column chromatography on silica gel using
as eluent dichloromethane–acetone 90:10 (v/v). Methyl,
ethyl and butyl 2-acetamido-3-(heteroaryl)propanoates
were prepared in the same manner.
3.27 (m, 2H), 4.43–4.49 (m, 1H), 6.60 (s, 1H), 7.15–7.23
(m, 2H), 7.47 (d, 1H), 7.53 (d, 1H), 8.08 (d, 1H, NH);
13C NMR (DMSO-d6): d = 22.5, 30.7, 51.6, 103.4, 110.6,
120.4, 122.5, 123.3, 128.5, 154.0, 156.0, 169.0, 172.9.
4.3.10.2.
rac-2-Acetamido-3-(benzo[b]thiophen-2-yl)-
propanoic acid rac-9b. Yield = 83%; mp 208 °C from
ethyl acetate–hexane (decomp.); HRMS: M+ found (M+
calculated for C13H13NO3S): 263.06191 (263.06161); MS:
m/z (%) = 263 (20.59. M), 206 (6.23), 205 (14.06), 204
(97.76), 187 (3.79), 176 (6.23), 163 (2.85), 162 (7.50), 161
(4.60), 160 (4.30), 154 (2.51), 153 (17.42), 151 (5.57), 150
(4.97), 149 (9.39), 148 (13.28), 147 (100.00), 136 (7.52),
135 (2.57), 134 (8.23), 121 (4.74), 116 (2.67), 115 (11.09),
108 (5.79), 107 (19.76), 106 (14.60), 105 (7.02), 104 (2.54),
103 (7.03), 102 (3.71), 91 (18.08), 90 (2.56); 1H NMR
(DMSO-d6): d = 1.84 (s, 3H), 3.18–3.39 (m, 2H), 4.47–
4.32 (m, 1H), 7.20 (s, 1H), 7.26–7.35 (m, 2H), 7.75 (d,
1H), 7.88 (d, 1H), 8.33 (d, 1H, NH); 13C NMR (DMSO-
d6): d = 22.5, 32.0, 53.2, 122.1, 122.7, 123.0, 123.8, 124.2,
139.0, 139.5, 141.0, 169.4, 172.5.
4.3.11.1. rac-Propyl 2-acetamido-3-(benzofuran-2-yl)-
propanoate rac-10a. Yield = 89%; semisolid, HRMS:
M+ found (M+ calculated for C16H19NO4): 289.13163
(289.13141); MS: m/z (%) = 291 (1.8, M+2), 290 (18,
M+1), 289 (100, M), 288 (17), 287 (12), 284 (11.5), 279
(20.5), 278 (20.5), 268 (11.3), 264 (20.8), 258 (27.1), 257
(15.2), 248 (58.4), 242 (17.3), 231 (18.5), 229 (12.4), 225
(12.7), 215 (12.3), 212 (13.6), 209 (14.9), 193 (22.1), 176
1
(12.2), 159 (20.1), 129 (14.6); H NMR (CDCl3): d = 0.92
(t, 3H), 1.60–1.74 (m, 2H), 1.99 (s, 3H), 3.33 (d, 2H),
4.05–4.18 (m, 2H), 4.92–4.98 (m, 1H), 6.47 (1H, NH),
6.49 (s,1H), 7.16–7.26 (m, 2H), 7.39 (d, 1H), 7.49 (d, 1H);
13C NMR (CDCl3): d = 10.3, 21.8, 23.0, 31.0, 51.2, 67.4,
104.9, 110.8, 120.6, 122.7, 123.8, 128.3, 153.5, 154.9,
170.0, 171.2.
4.3.10.3. rac-2-Acetamido-3-(benzofuran-3-yl)propanoic
acid rac-9c. Yield = 86%; mp 156 °C from ethanol–water
(155–156 °C from water21); HRMS: M+ found (M+ calcu-
lated for C13H13NO4): 247.08531 (247.08446); MS: m/z
(%) = 249 (0.49, M+2), 248 (4.27, M+1), 247 (29.44, M),
232 (0.25), 229 (0.87), 216 (0.79), 206 (0.26), 205 (0.72),
204 (3.99), 203 (1.60), 202 (0.55), 201 (0.65), 190 (1.03),
189 (10.04), 188 (100.00), 187 (3.50), 186 (0.54), 185
(0.37), 172 (0.39), 171 (2.19), 170 (0.24), 162 (0.32), 161
(1.07), 160 (8.72), 159 (4.34), 158 (1.60), 149 (0.28), 148
(0.38), 147 (3.17), 146 (25.27), 145 (2.05), 144 (6.47), 143
(0.37), 142 (0.55), 134 (0.52), 133 (1.23), 132 (8.96), 131
(79.93), 130 (1.62), 121 (0.74), 119 (0.31), 118 (2.73), 117
(0.45), 116 (0.71), 115 (4.11), 114 (0.39), 105 (1.18), 104
4.3.11.2. rac-Propyl 2-acetamido-3-(benzo[b]-thiophen-2-
yl)propanoate rac-10b. Yield = 88%; semisolid, HRMS:
M+ found (M+ calculated for C16H19NO3S): 305.10796
(305.10856); MS: m/z (%) = 307 (9.8, M+2), 306 (23.1,
M+1), 305 (100, M), 304 (32.9), 303 (31.6), 302 (13.9),
301 (14.0), 300 (35.6), 290 (64.1), 289 (69.6), 288 (33.4),
287 (36.8), 286 (39.1), 285 (16.2), 275 (20.1), 271 (17.4),
270 (70.4), 260 (16.6), 257 (15.4), 256 (26.7), 247 (16.6),
225 (24.4), 219 (15.2), 213 (23.5), 201 (8.9), 185 (11.3),
1
(1.09), 103 (7.21), 102 (1.78), 101 (0.24), 91.1 (0.49); H
1
NMR (DMSO-d6): d = 1.86 (s, 3H), 3.01–3.21 (m, 2H),
4.35–4.61 (m, 1H), 7.30–7.39 (m, 2H), 7.60 (d, 2H), 7.71
(d, 1H), 7.81 (s, 1H), 8.33 (d, 1H, NH); 13C NMR
(DMSO-d6): d = 22.3, 25.3, 51.8, 111.2, 116.0, 119.6,
122.5, 124.2, 127.7, 142.9, 154.4, 169.3, 173.9.
170(6.7), 144 (2.3), 127 (6.8); H NMR(300 MHz, CDCl3):
d = 0.94 (t, 3H), 1.63–1.74 (m, 2H), 2.03 (s, 3H), 3.38–3.52
(m, 2H), 4.12 (t, 2H), 4.92–4.98 (m, 1H), 6.43 (1H, NH),
7.01 (s, 1H), 7.25–7.35 (m, 2H), 7.69 (d, 1H), 7.76 (d,
1H); 13C NMR (75 MHz, CDCl3): d = 10.3, 21.8, 23.1,

P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
509
32.8, 52.8, 67.4, 122.1, 123.0, 123.4, 124.0, 124.3, 138.6,
139.7, 139.8, 169.9, 171.0.
HRMS: M+ found (M+ calculated for C11H11NO3):
205.07366 (205.07389); MS: m/z (%) = 206 (5.14, M+1),
205 (30.38, M), 160 (5.34), 159 (4.48), 149 (2.42), 148
(3.31), 147 (16.70), 144 (2.54), 133 (5.48), 132 (41.90), 131
(100), 130 (3.62), 115 (5.96), 105 (5.59), 104 (2.73), 103
(9.94), 102 (6.74), 89 (6.14); 1H NMR (DMSO-d6):
d = 3.43 (d, 2H), 4.27 (t, 1H), 6.79 (s, 1H), 7.20–7.29 (m,
2H), 7.50 (d, 1H), 7.59 (d, 1H), 8.70 (s, 3H, NH3þ); 13C
NMR (DMSO-d6): d = 28.8, 50.7, 105.7, 110.9, 120.8,
122.7, 123.9, 128.2, 152.1, 154.4, 169.8.
4.3.11.3. rac-Propyl 2-acetamido-3-(benzofuran-3-yl)pro-
panoate rac-10c. Yield = 91%; semisolid, HRMS: M+
found (M+ calculated for C16H19NO4): 289.13158
(289.13141); MS: m/z (%) = 291 (1.8, M+2), 290 (18,
M+1), 289 (100, M), 288 (57), 287 (35), 284 (17.1), 280
(11.2), 279 (33.5), 278 (35.6), 277 (44.1), 276 (24.8), 264
(19.2), 259 (12.2), 254 (13.9), 252 (16.2), 249 (29.4), 248
(48.1), 247 (65.7), 246 (98), 219 (17.6), 202 (14.5), 135
1
(18.5), 129 (14.8); H NMR (300 MHz, CDCl3): d = 0.87
4.3.13.2. rac-2-Amino-3-(benzo[b]thiophen-2-yl)-propa-
noic acid rac-12b. Yield = 78%; mp 258 °C from water–
ethanol (258 °C from water–ethanol22); HRMS: M+ found
(M+ calculated for C11H11NO2S): 221.05089 (221.05105);
MS: m/z (%) = 222 (2.05, M+1), 221 (12.61, M), 204
(0.92), 176 (2.72), 175 (1.49), 174 (0.78), 165 (1.85), 160
(1.13), 150 (2.48), 149 (8.44), 148 (27.07), 147 (100.00),
146 (1.69), 145 (2.25), 143 (1.76), 134 (4.91), 121 (3.03),
116 (1.64), 115 (8.00), 114 (1.11), 108 (0.98), 104 (1.00),
103 (5.37), 102 (2.87), 101 (1.17), 95 (1.10); 1H NMR
(DMSO-d6): d = 3.45–3.58 (m, 2H), 4.28 (t, 1H), 7.31–
7.39 (m, 3H), 7.80 (d, 1H), 7.93 (d, 1H), 8.57 (s, 1H,
NH3þ); 13C NMR (DMSO-d6): d = 30.6, 52.6, 122.2,
123.3, 124.2, 124.4, 124.7, 137.2, 139.4, 139.5, 169.8.
(t, 3H), 1.54–1.66 (m, 2H), 1.98 (s, 3H), 3.15–3.30 (m,
2H), 3.96–4.11 (m, 2H), 4.92–4.98 (m, 1H), 6.40 (1H,
NH), 7.21–7.32 (m, 2H), 7.42–7.53 (m, 3H); 13C NMR
(75 MHz, CDCl3): d = 10.2, 21.7, 23.1, 26.2, 52.3, 67.3,
111.5, 114.9, 119.4, 122.6, 124.5, 128.0, 142.4, 155.1,
169.9, 171.7.
4.3.11.4. rac-Propyl 2-acetamido-3-(benzo[b]-thiophen-3-
yl)propanoate rac-10d. Yield = 84%; semisolid, HRMS:
M+ found (M+ calculated for C16H19NO3S): 305.10879
(305.10856); MS: m/z (%) = 307 (9.8, M+2), 306 (23.1,
M+1), 305 (100, M), 304 (47.2), 303 (13.4), 289 (2.3), 269
(3.3), 265 (5.2), 264 (4.9), 263 (12.2), 261 (1.4), 246 (3.0),
245 (7.5), 233 (1.9), 226 (2.3), 222 (5.9), 221 (9.1), 220
(1.8), 217 (2.7), 214 (2.1), 211 (4.6), 150 (2.0), 123 (5.3);
1H NMR(300 MHz, CDCl3): d = 0.85 (t, 3H), 1.53–
1.60(m, 2H), 1.95 (s, 3H), 3.31–3.45 (m, 2H), 3.92–4.09
(m, 2H), 4.96–5.02 (m, 1H), 6.40 (1H, NH), 7.15 (s, 1H),
7.31–7.41 (m, 2H), 7.76 (d, 1H), 7.85 (d, 1H); 13C NMR
(75 MHz, CDCl3): d = 10.2, 21.7, 23.0, 30.8, 52.5, 67.2,
121.5, 122.8, 123.7, 124.1, 124.4, 130.8, 138.9, 140.2,
170.0, 171.8.
4.3.13.3. rac-2-Amino-3-(benzofuran-3-yl)propanoic acid
rac-12c. Yield = 92%; mp 240 °C from water (240 °C
from water20); HRMS: M+ found (M+ calculated for
C11H11NO3): 205.07415 (205.07389); MS: m/z (%) = 206
(2.60, M+1), 205 (16.11, M), 160 (5.37), 133 (3.82), 132
(41.68), 131 (100.00), 130 (5.95), 118 (0.65), 115 (12.99),
107 (1.99), 106 (0.73), 105 (6.78), 104 (6.70), 103 (31.70),
1
102 (11.02), 101 (1.55), 98 (1.80); H NMR (DMSO-d6):
d = 3.30 (d, 2H), 4.22 (t, 1H), 7.26–7.36 (m, 2H), 7.58 (d,
1H), 7.72 (d, 1H), 7.88 (s, 1H), 8.56 (s, 3H, NH3þ); 13C
NMR (DMSO-d6): d = 24.2, 51.5, 111.3, 113.4, 119.9,
122.6, 124.3, 127.3, 144.3, 154.6, 170.4.
4.3.12. Synthesis of rac-4-((heteroaryl)methyl)-2-meth-
yloxazol-5(4H)-ones rac-11a–d. Into a solution of rac-2-
acetamido-3-(heteroaryl)propanoic acid rac-9a–d (0.5
mmol) in anhydrous dichloromethane (5 mL), a solution
of dicyclohexylcarbodiimide (135 mg, 0.6 mmol) in anhy-
drous dichloromethane (2 mL) was added dropwise at
0 °C. Before the addition was completed, 1,3-dicyclohexyl-
urea started to precipitate. The reaction mixture was stirred
further for 5–10 min. At the end, the solid was filtered off
and dichloromethane was removed under reduced pressure
at room temperature yielding the desired product. The
obtained rac-4-((heteroaryl)methyl)-2-methyloxazol-5(4H)-
one rac-11a–d was immediately used in the enzymatic reac-
tions without further purification.
4.3.13.4. rac-2-Amino-3-(benzo[b]thiophen-3-yl)-propa-
noic acid rac-12d. Yield = 75%; mp 280 °C from water
(279–280 °C23); HRMS: M+ found (M+ calculated for
C11H11NO2S): 221.05133 (221.05105); MS: m/z (%) = 222
(2.55, M+1), 221 (15.82, M), 185 (4.76), 178 (2.08), 177
(1.58), 176 (4.81), 175 (6.03), 174 (4.02), 173 (2.11), 172
(2.38), 157 (1.31), 153 (1.25), 150 (1.79), 149 (8.52), 148
(24.05), 147 (100), 146 (1.52), 145 (2.38), 144 (1.36), 143
(3.20), 134 (4.08), 131 (1.19), 129 (1.99), 128 (1.97), 127
(1.61), 121 (1.95), 116 (1.85), 115 (7.48), 114 (1.83), 103
(5.49), 102 (2.41), 101 (1.15), 98 (1.38); 1H NMR
(DMSO-d6): d = 3.42 (d, 2H), 4.14 (t, 1H), 7.38–7.45 (m,
2H), 7.64 (s, 1H), 7.93 (d, 1H), 8.00 (d, 1H), 8.58 (s, 3H,
NH3þ); 13C NMR (DMSO-d6): d = 28.9, 51.9, 121.7,
122.9, 124.2, 124.4, 125.8, 129.5, 138.3, 139.7, 170.3.
4.3.13. Synthesis of rac-2-amino-3-(heteroaryl)-propanoic
acids rac-12a–d. The rac-2-acetamido-3-(heteroaryl)prop-
anoic acid 9a-d (1 g) was suspended in half concentrated
HCl (10 mL) and the mixtures were refluxed for 4 h, cooled
to room temperature, obtaining the product as a white pre-
cipitate, which was filtered, dried and finally washed with
diethyl ether. The precipitate was resuspended in water
and the pH was adjusted to 5.6, stirred for 30 min, filtered
and dried at room temperature.
4.4. Enzymatic small scale reactions
4.4.1. Dynamic kinetic resolution of rac-4-((hetero-
aryl)methyl)-2-methyloxazol-5(4H)-ones rac-11a–d. In a
typical small scale experiment, lipase (20 mg) was added
4.3.13.1. rac-2-Amino-3-(benzofuran-2-yl)propanoic acid
rac-12a. Yield = 93%; mp 205 °C from water–ethanol;
to
a
solution of rac-4-((heteroaryl)methyl)-2-meth-
yloxazol-5(4H)-one rac-11a–d (20 mg) in different alcohols

510
P. V. Podea et al. / Tetrahedron: Asymmetry 19 (2008) 500–511
(methanol, ethanol, 1-propanol, 1-butanol) (1 mL) and the
mixture was stirred at the room temperature. Samples
(10 lL) were taken at intervals, diluted with hexane–iso-
propyl alcohol (9:1) (990 lL) and analyzed by HPLC using
a Chiralpak IA column.
suspended into a vigorously stirred solution of sodium car-
bonate (0.053 g, 5 mmol) in water (8 mL) and the mixture
was gently refluxed. After 2 h of heating, the solution was
cooled to 5 °C and extracted with dichloromethane
(3 ꢂ 10 mL). The aqueous phase was then acidified care-
fully with concentrated HCl solution. The deposited pre-
cipitate was filtered off and washed several times with
cold water. The isolated L-2-acetamido-3-(heteroaryl)prop-
anoic acid L-9a–d was then dissolved in water (10 mL) by
adjusting the pH to 8 with LiOH solution (1.25 M). Then
Acylase I (22 units, 60 mg) and CoCl2 ꢂ 6H2O (50 mg
0.2 mmol) were added and the reaction mixture was stirred
at 37 °C, while by the addition of LiOH solution (1.25 M),
the pH of the solution was kept between 7 and 8. After
completion of the L-2-acetamido-3-(heteroaryl)-propanoic
acid L-9a–d hydrolysis, the pH was adjusted to 1.5 with
5% HCl. The suspension formed was extracted with dichlo-
romethane (3 ꢂ 10 mL). The aqueous phase was heated
with active charcoal (50 mg) to 90 °C for 2 min, cooled at
room temperature, filtered and applied to a Dowex 50X8
cation exchange resin column. Elution of the pure L-enan-
tiomers of the 2-amino-3-(heteroaryl)propanoic acids L-
12a–d occurred with 2 M ammonia solution.
For improving the ee, different solvents were used as reac-
tion media.
In a typical small scale experiment, Novozyme 435 (20 mg)
was added into a solution of rac-4-((heteroaryl)methyl)-2-
methyloxazol-5(4H)-one rac-11a–d (20 mg) and 2 equiva-
lents of propanol in different kinds of solvents (1 mL) were
added. The mixture was stirred at room temperature and
samples (10 lL) were taken, diluted with hexane–isopropyl
alcohol (9:1) (990 lL) and analyzed by HPLC using a Chir-
alpak IA column.
4.4.2. Kinetic resolution of rac-2-acetamido-3-(hetero-
aryl)propanoic acids rac-9a–d. rac-2-Acetamido-3-(het-
eroaryl)propanoic acid rac-9a-d (1 mmol) was suspended
in water (10 mL). By adjusting the pH to 8 with LiOH solu-
tion (1.25 M), the suspension merged into solution. Then
Acylase I (5 units, 12 mg) and CoCl2 ꢂ 6H2O (5 mg,
0.02 mmol) were added and the reaction mixture was stir-
red at 37 °C, while by the addition of LiOH solution
(1.25 M), the pH of the solution was permanently kept
between 7 and 8. After completion of L-2-acetamido-
3-(heteroaryl)propanoic acid L-9a–d hydrolysis (approx.
24 h, checked by HPLC using an Astec Chirobiotic-Tag
column with methanol and TEAA-buffer, pH 4.1, 80:20
(v/v) as eluent) the pH was adjusted to 1.5 with 5% HCl.
The untransformed enantiopure D-2-acetamido-3-(hetero-
aryl)propanoic acid D-9a–d was filtered off and washed
with deionized water (3 ꢂ 1 mL). The filtrate was heated
with active charcoal (10 mg) to 90 °C for 2 min, cooled to
room temperature, filtered, and applied to a Dowex 50X8
cation exchange resin column. Elution of the pure L-enan-
tiomer of the 2-amino-3-(heteroaryl)propanoic acid L-12a–
d occurred with 2 M ammonia solution.
Acknowledgements
Financial support from the Romanian Ministry of Educa-
tion and Research (CEEX Contract No. 1480-6/
07.04.2006) is gratefully acknowledged.
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